The Human Immunodeficiency Virus (HIV) is the causative agent of Acquired Immunodeficiency Syndrome, a devastating infection that has reached pandemic proportions. The first and pre- requisite step for HIV entry into a permissive cell is specific binding of the viral glycoprotein gp120 to the cellular CD4 receptor;this interaction triggers an ordered series of steps that ultimately results in fusion of the viral envelope with the cellular membrane and entry of the nucleocapsid into the cell interior. Gp120 is tethered to the viral envelope via non-covalent interactions with trimeric gp41, an integral membrane protein that mediates membrane fusion events. Trimeric gp120/gp41 spikes represent the primary epitope exposed on the viral surface and a variety of trimeric "gp140" deletion constructs and soluble monomeric gp120 constructs have been studied as immunogens to elicit broadly neutralizing antibodies (bNtAbs) to HIV. Despite heroic efforts, induction of bNtAbs against primary isolates of HIV has been uniformly unsuccessful. This has led to the conclusion that a fundamental understanding of the HIV envelope glycoprotein (Env) spike structure and its interaction with bNtAbs is key to future vaccine development. Unfortunately, this goal has been frustrated by the lack of an in vitro system that allows the study of Env trimers in a soluble, biologically relevant, and functional lipid-bound conformation. Phospholipid nanodiscs provide such a system. Nanodiscs are derived from high-density lipoprotein particles involved in reverse cholesterol transport in humans;they provide stable model membranes into which membrane proteins can be embedded in a native and functional form. They are homogenous in size and defined in composition, and provide a stable, soluble, and mono-disperse platform that is amenable to rigorous biochemical, biophysical, and structural interrogation. We propose to utilize nanodisc technology to assemble HIV Env trimers in a defined lipid bilayer and to characterize the structure and function of the trimeric spikes. This application represents a "proof-of-concept" proposal that is ideally suited to an R21 application as it represents modest-risk, high yield proposal. Successful completion of the stated goals will provide a novel platform to study Env structure and function and provide the foundation for future studies that will utilize this innovative immunogen for HIV vaccine development. PUBLIC HEALTH RELEVANCE: HIV infection has reached pandemic proportions and the development of an effective vaccine will be paramount to containing AIDS. Previous attempts at vaccine development have been disappointing, in large part because the "rationally designed" immunogens have failed to mimic the natural presentation of the HIV envelope (Env) spike structure. We propose to utilize nanodisc technology to assemble and characterize Env spikes in a soluble lipid bilayer. This work will provide the foundation for future studies that will utilize this innovative immunogen for HIV vaccine development.